Effects of bifemelane hydrochloride (MCI-2016) on acetylcholine (ACh) level in the cerebral cortex and hippocampus of rats and Mongolian gerbils were examined. In normal rats, MCI-2016 (30 mg/kg, i.p.) slightly increased ACh content in the cerebral cortex. Scopolamine (1 mg/kg, i.p.) or hypoxia (95% N2 +5% O2, 9 min) decreased ACh level and pretreatment of MCI-2016 attenuated the decrement of ACh level in the rats. ACh level in the brain of Mongolian gerbils was significantly decreased following ligation of bilateral carotid arteries. In this case, MCI-2016 also attenuated the decrement of ACh level. These results suggest that improvement by MCI-2016 of behavioral impairment observed in the animals treated with scopolamine, hypoxia or ischemia may be, at least partly, attributed to the amelioration of decreased ACh level in the brain.